REVIEWS
Maintaining genome stability at the
replication fork
Dana Branzei*and Marco Foiani*‡
Abstract | Aberrant DNA replication is a major source of the mutations and chromosome
rearrangements that are associated with pathological disorders. When replication is
compromised, DNA becomes more prone to breakage. Secondary structures, highly
transcribed DNA sequences and damaged DNA stall replication forks, which then require
checkpoint factors and specialized enzymatic activities for their stabilization and subsequent
advance. These mechanisms ensure that the local DNA damage response, which enables
replication fork progression and DNA repair in S phase, is coupled with cell cycle transitions.
The mechanisms that operate in eukaryotic cells to promote replication fork integrity and
coordinate replication with other aspects of chromosome maintenance are becoming clear.
Autonomously replicating
sequence
A DNA element in the yeast
genome that contains origins
of replication.
Replication fork
The branch point structure that
forms during DNA replication
between two template DNA
strands, at which nascent
DNA synthesis is ongoing.
*Fondazione IFOM,
Istituto FIRC di Oncologia
Molecolare, IFOM-IEO
campus, Via Adamello 16,
20139 Milan, Italy.
‡
Dipartimento di Scienze
Biomolecolari e Biotecnologie,
Università degli Studi di
Milano, Via Celoria 26,
20133 Milan, Italy.
e-mails: [email protected]; marco.foiani@
ifom-ieo-campus.it
doi:10.1038/nrm2852
Eukaryotic cells regulate the replication of their genomes
in a highly complex manner that ensures the accurate
and speedy duplication of the genetic information and
preserves genome stability. DNA replication is tightly
monitored to ensure that the genome is replicated just
once per cell cycle, and that DNA replication is complete before mitosis begins. The sequence of events and
many of the proteins involved in DNA replication are
conserved throughout the eukaryotic world, although
there are differences in the complexity of factors and in
the mechanisms regulating DNA replication in different
eukaryotes1,2.
Replication initiates from multiple regions distributed
along chromosomes (FIG. 1a). Certain organisms, such as
the budding yeast Saccharomyces cerevisiae, have clear replicator sequences called autonomously replicating sequences
(ARSs), whereas many other organisms have more relaxed
DNA sequence requirements for the initiation events3. In
S. cerevisiae, analysis of the replication time of all chromosomal segments suggests that origins differ in their time of
initiation or ‘firing’, from early to mid and late S phase, as
well as in their firing efficiency 4–6. Furthermore, the findings that there are more ARSs — which represent potential
origins — than active origins on chromosomes, and that
there is no phenotype associated with deleting replication
origins, suggest that origins might be present in excess
and used flexibly, with the passively replicated dormant
origins being fired when major ones are inactivated1,6,7.
All organisms studied so far seem to have an excess of
origins3,6,8–10. The origins are marked by the formation
of a pre-replicative complex (preRC) in G1, before DNA
replication, through the binding of the origin recognition
complex (ORC) and the recruitment of additional replication factors, such as cell division control protein 6 (Cdc6),
chromatin licensing and DNA replication factor 1 (Cdt1)
and the minichromosome maintenance (MCM) helicase
complex, which contains the six subunits Mcm2–Mcm7,
to these sites. Because the preRC cannot be assembled
later in the cell cycle, owing to the inhibitory activity
of the S, G2 and M phase cyclin-dependent kinases (CDKs),
the maximum number of origins available for an S phase
is determined during the licensing state, which occurs
in G1 when the preRC is formed. Furthermore, replication through an origin must remove or inhibit preRCs in
order to prevent re-replication3,11. At each fired origin, two
sister replication forks (RFs) are established that move away
from the origin as the parental DNA duplex is unwound
by the action of DNA helicases (FIG. 1b). The RFs can be
hindered by physical impediments such as protein–DNA
complexes and DNA damage. Different factors, such as
the S. cerevisiae helicase Rrm3 (FIG. 1c)12 and proteins at the
replication checkpoint13 (BOX 1), act to ensure the progression of RFs through natural fragile regions. When one RF
is terminally blocked or arrested, firing of dormant or
nearby origins ensures that replication is complete.
DNA replication is an amazing undertaking as cells
need to monitor the integrity of RFs while coupling replication with other cellular processes, such as chromatin
reassembly, the inheritance of the epigenetic chromatin
structure and the establishment of cohesion between
sister chromatids. Cells replicating their DNA must also
be able to initiate an adequate DNA damage response
(DDR), execute DNA repair and replicate chromosome
regions such as chromosome ends (telomeres) and
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a
preRC
preRC
MCM
Origin of replication
Origin of replication
b
MCM
MCM
Leading strand
MCM
Discontinuous
lagging strand
Fired origin
Leading strand
c
Top2
RPA
LigI
Polε
Rad27
Mrc1
MCM
RNase H
Polα
Dna2 Tof1
Csm3 Rrm3
RFC
Polδ
PCNA
Top1
Figure 1 | Replication initiation and progression. a | Replication begins from multiple
Nature Reviews | Molecular Cell Biology
origins, which are marked by the formation of a pre-replicative complex (preRC).
b | Two replication forks (RFs), which are associated with the replisome that carries out
DNA replication, are established at each fired origin. The minichromosome maintenance
(MCM) helicase complex is shown ahead of the RFs, unwinding the duplex DNA.
Replication is semidiscontinuous: DNA synthesis is continuous on the leading strand and
discontinuous on the lagging strand, on which primers are elongated to form Okazaki
fragments that are processed and ligated to one another. c | Numerous proteins are
present at the RF. The MCM helicase unwinds the parental duplex, allowing access to the
DNA polymerase-α (Polα) primase, replicative polymerase-δ (Polδ) and polymerase-ε
(Polε) (which elongate the primers) and the replication processivity clamp proliferating
cell nuclear antigen (PCNA; also known as Pol30), which is loaded by the clamp loader,
the replication factor C (RFC) complex. Replication protein A (RPA) binds single-stranded
DNA regions exposed at the RF or during lagging-strand synthesis. The discontinuous
fragments synthesized on the lagging strand are processed by Rad27 (FEN1 in humans),
Dna2 helicase, RNase H, Polδ and DNA ligase I (LigI). Several other factors associate
with the RF in yeast and are represented: DNA topoisomerases 1 (Top1) and Top2,
the checkpoint mediators mediator of replication checkpoint protein 1 (Mrc1),
Top1-associated factor 1 (Tof1) and chromosome segregation in meiosis protein 3
(Csm3), and the Rrm3 helicase.
GINS complex
An essential complex for DNA
replication that promotes
polymerase-ε loading and the
activity of the MCM helicase.
ribosomal DNA containing repetitive DNA regions14–18.
Several enzymatic activities in addition to the ones
required for replication per se (FIG. 1c) are associated
with the RF. These include checkpoint mediators that
prevent the collapse of occasionally stalled RFs (BOX 1),
nucleosome assembly and remodelling factors, DNA
topoisomerases and factors required to establish sister
chromatid cohesion.
Genetic mutations that affect enzymes involved in
replication result in an accumulation of DNA intermediates that trigger recombination and genome instability 19,20. Given the complexity of the tasks that must be
completed during replication, and their need to be coordinated with one another, it is not surprising that defects
in DNA replication or its regulation underpin many
human diseases and ageing. Therefore, understanding
DNA replication and the pathways that suppress the
instability of RFs is directly relevant to understanding
the mechanisms by which cancers and other pathological
disorders arise.
here, we review the main causes of RF stalling and
collapse, and the pathways that prevent RF instability. We
begin with an overview of the basic aspects of RF progression and the topological mechanisms that assist DNA
metabolism during DNA replication. We discuss the most
important elements that affect RF integrity and how they
can lead to a source of DNA breaks, and we explain the
functions of checkpoints (BOX 1), DNA topoisomerases
(BOX 2) and other specialized factors in maintaining RF
stability and in preventing aberrant DNA transitions.
Next, we consider the specialized pathways that deal
with the distinct constraints imposed on DNA by different types of lesions and how they ensure RF progression.
Finally, we comment on the consequences of treating
genomic instability disorders with checkpoint or topoisomerase inhibitors, and on how the knowledge derived
from genetic studies could reveal potential gene targets for
disease prevention and therapy.
RF dynamics
unwinding of the DNA is as essential a step for DNA
replication initiation as it is for elongation. Formation
of an active helicase at replication origins involves,
besides preRC assembly, the recruitment of factors such
as Cdc45 (which is essential for DNA replication initiation and travels with the RF) and the GINS complex,
which interact with the core of the MCM replicative
helicase complex, Mcm2–Mcm7 (ReFS 21,22). Origin
firing also requires the activity of the S phase CDK
and the Cdc7–Dbf4 kinase complex (also known as
Dbf4-dependent kinase (DDK))23–26. The GINS complex is also important for the progression of RFs as it
maintains the association between MCM and Cdc45
within the replisome27. The replisome is a multicomponent protein complex that is associated with the
RF. It includes the MCM replicative helicase complex,
replication elongation factors such as DNA polymerases (polymerase-α, polymerase-ε and polymerase-δ)
and polymerase accessory factors (the clamp loader
replication factor C (RFC) and the clamp proliferating cell nuclear antigen (PCNA; also known as Pol30
in S.cerevisiae)), mediator of replication checkpoint
protein 1 (Mrc1) and the topoisomerase 1-associated
factor 1 (Tof1)–chromosome segregation in meiosis
protein 3 (Csm3) complex that is required for RF
pausing (FIG. 1c). Since the chemistry of DNA synthesis
dictates that the nucleotides are added to the 3′ end
of the primer synthesized by the polymerase-α
primase, the replication is semi-discontinuous: there
is a leading strand, on which DNA nucleotides are
added continuously to the initial primer, and a lagging
strand, on which primers are synthesized and elongated
throughout DNA synthesis to form Okazaki fragments.
Once formed, Okazaki fragments are processed and
ligated to the previous fragment (FIG. 1b).
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below), which are required to activate a robust DDR, the
ubiquitylation pathway also responds to different types of
defects that arise during DNA replication and contributes
to the replication stress response (BOX 3).
Box 1 | The damage and replication checkpoint
Replication stress or DNA damage
Stop
Stalled RF
ATR (or Mec1)
Terminal RF
DSB
ATM (or Tel1) and
MRN (or MRX)
EXO1-mediated
processing
EXO1-mediated
processing
Checkpoints are cellular surveillance and signalling Nature
pathways
that detect
DNACell
lesions,
Reviews
| Molecular
Biology
such as single-stranded (ss)DNA or double-strand breaks (DSBs), and then boost a DNA
damage response that ensures replication fork (RF) stabilization, DNA repair and cell
cycle arrest. Ataxia telangiectasia mutated (ATM; telomere length regulation protein 1
(Tel1) in yeast) and ATM- and Rad3-related protein (ATR; mitosis entry checkpoint
protein 1 (Mec1) in yeast) are key players in triggering the S phase checkpoint response.
ATM (or Tel1) responds mostly to DSBs and is recruited to these through the MRN
complex, which is composed of MRE11, RAD50 and NBS1 (or the MRX complex in yeast,
which is composed of Mre11, Rad50 and Xrs2). ATM (or Tel1) and MRN (or MRX) prevent
reversed RF formation and the formation of recombination structures derived from RFs
that encounter DSBs (terminal RFs75; see the figure). ATR (or Mec1), however, responds
mostly to stalled RFs with exposed ssDNA coated with replication protein A (RPA) to
promote its stabilization (see the figure). In replication checkpoint mutants, the
replisome dissociates from DNA29,30, leading to the formation of reversed RFs80 that
are processed by exodeoxyribonuclease 1 (EXO1)82, which leads to long stretches of
ssDNA73,74,83 (see the figure). The ssDNA–RPA that forms at stalled RFs acts to recruit two
checkpoint complexes: ATR–ATR-interacting protein (ATRIP; Mec1–Ddc2 in yeast) and
the checkpoint clamp loader RAD17 (Rad24 in yeast), which is required to load the
proliferating cell nuclear antigen (PCNA)-like checkpoint clamp RAD9–RAD1–HUS1
(also known as 9-1-1; Rad17–Mec3–Ddc1 in yeast) onto ssDNA. 9-1-1 is then further
phosphorylated by ATR (or Mec1) and this ensures robust checkpoint activation.
Following ATM (or Tel1) and ATR (or Mec1) activation, mediator proteins are recruited
to the site of DNA damage, and through phosphorylation of the effector checkpoint
kinases CHK2 (also known as CHEK2) and CHK1 (also known as CHEK1), respectively,
they act to amplify the checkpoint signal throughout the cell141,144. The functional
orthologue of CHK1 in yeast is Rad53 but in yeast, following DNA damage, Mec1
activates both Rad53 and Chk1. The roles of Rad53 in stabilizing RFs are well
documented29,30,73,74,83; Chk1 also appears to affect replication through damaged DNA145.
The activation of the effector kinases triggers the checkpoint response through the
phosphorylation of targets that are implicated in different, specific processes141,144.
Precatenane
A cruciform junction that is
formed by the intertwining of
sister duplexes in the replicated
portion of a replicon.
The progression of individual RFs can be stopped for
various reasons, such as the presence of protein–DNA
complexes or depletion of the nucleotide pool. In S. cerevisiae, proteins such as Mrc1 and Tof1 limit progression
of the replisome under such circumstances28 and the
checkpoint kinases mitosis entry checkpoint protein 1
(Mec1) and Rad53 (BOX 1) stabilize stalled RFs and prevent disassembly of the replisome29,30, thus preventing RF
collapse. In addition to checkpoint proteins (BOX 1 and see
DNA topology
unwinding of the duplex DNA induces differences in
the topology and conformation of replicating DNA. The
intertwining of the complementary strands in any DNA
molecule is described by the linking number, which measures the number of times that one strand crosses the other
strand in the DNA helix, and the number of times that one
segment of double helix crosses another in higher-order
superhelical structures31. The torsional stress created by the
replication of any DNA segment cannot simply diffuse by
the swivelling of the extremities of chromosomes because
eukaryotic chromosomes are very large. Therefore,
this tension can only be altered by DNA breakage and
reunion reactions, which are mediated by specialized
DNA nucleases called topoisomerases32 (BOX 2).
Separation of the parental DNA strands by DNA helicases locally reduces the linking number of DNA molecules, generating compensatory positive torsional stress
that can take the form of either supercoiling ahead of
the RF or precatenanes that intertwine the two replicated
duplexes behind the RF by swivelling the DNA at the RF
branch point (FIG. 2a). Positive supercoils ahead of RFs can
be removed in eukaryotes by type IB (TOP1) and type II
(TOP2) topoisomerases (BOX 2), and this sustains the progression of the replication machinery (FIG. 2a). Indeed,
both Top1 and Top2 were shown to travel with RFs in
S. cerevisiae33. Precatenanes, however, do not affect helix
unwinding and progression of the replication machinery.
In fact, their formation diffuses the positive superhelical
stress that accumulates in front of the RF. however, as precatenanes wind the daughter chromosomes around each
other, they oppose partitioning of the chromosomes and
therefore must be removed by topoisomerases. Catenanes
and precatenanes can only be removed by the action of
type II topoisomerases (BOX 2), which mediate the passage of an entire duplex through the other duplex that is
present at the sites of sister chromatid juxtaposition32.
Additional topological problems are generated when
two RFs fuse at termination (FIG. 2b). The length of the
unreplicated DNA becomes shorter as the RFs approach
one another, such that the space that can contain the positive supercoils that are expected to result from RF movement becomes limited and RFs must rely on precatenane
formation behind them in order to progress34 (FIG. 2b). The
fusion of the two RFs will finally give rise to catenated
sister chromatid junctions that have to be resolved before
DNA segregation. limited Top1 and Top2 activity, and
the increased requirements for DNA breakage–sealing
reactions at catenated sister chromatid junctions, might
explain the observation that late replicating regions are
prone to breakage and exhibit fragility 13,35,36.
Another situation that occurs during DNA synthesis
is that RFs encounter transcription bubbles (FIG. 2c). Both
head-on and in-line collisions between the replisome
and the RNA polymerase (which is associated with the
transcription bubble) have been shown to slow down
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Box 2 | DNA transitions mediated by DNA topoisomerases
Topoisomerases are specialized enzymes that catalyze the passage of DNA single strands
or double helices through one another (reviewed in ReFS 32,146). These transitions are
mediated by transesterification reactions. The topoisomerase first becomes covalently
attached to one of the ends of the DNA strand that has been attacked by the enzyme
through a phosphotyrosine linkage. The hydroxyl group from one broken DNA end then
attacks the phosphorus of the phosphotyrosine link, releasing the topoisomerase and
rejoining the DNA strands. There are two general types of topoisomerases. Type I
enzymes make single-strand breaks, allowing the uncut strand to pass through the break
before resealing the nick. Type II enzymes are dimeric and coordinately introduce
transient double-strand breaks in a DNA double helix, through which they then pass a
segment of uncut DNA before resealing the break. Type II topoisomerases require ATP
hydrolysis for their action, whereas type I topoisomerases do not. The energy released
through ATP hydrolysis is used to promote topological transformations rather than to
cleave or rejoin DNA.
The two types of topoisomerase can be further divided into subfamilies (see the table):
type IA (Top3), type IB (Top1), and type IIA (Top2). Type IA topoisomerases act to relax
negatively supercoiled DNA and to pass one double helix through another if at least one
of them contains a nick or a gap. The type IA topoisomerase, Top3, was proposed to act
in concert with the RecQ helicases Sgs1 in yeast and Bloom syndrome protein (BLM) in
humans to promote the resolution of double Holliday junctions91,110. Top1 enzymes are
very efficient at relaxing both positively and negatively supercoiled DNA and probably
provide the swivels for replication fork movement during replication by removing
the positive supercoils in the unreplicated region. Top2 is required to decatenate
chromosomes and precatenanes, and can relax both positive and negative supercoils,
perhaps substituting for Top1 in its absence.
Topoisomerase
subfamily
Representative eukaryotic member
Type IA
Yeast topoisomerase 3 (Top3)
Mammalian topoisomerase 3α (TOP3A) and 3β (TOP3B)
Type IB
Yeast topoisomerase 1 (Top1)
Mammalian topoisomerase 1 (TOP1)
Mammalian mitochondrial topoisomerase 1
Type IIA
Yeast topoisomerase 2 (Top2)
Mammalian topoisomerase 2α (TOP2A) and 2β (TOP2B)
Supercoil
A contortion in DNA that is
important for DNA packaging
and DNA and RNA synthesis.
Topoisomerases sense
supercoiling and act to either
generate or dissipate it by
changing DNA topology.
Catenane
An interlocked DNA molecule.
Late replication zone
A DNA region that replicates
late during S phase.
Triplex H‑DNA
A DNA structure in which a
DNA duplex associates with
another DNA single strand,
in either a parallel or
antiparallel orientation.
Left‑handed Z‑DNA
One of the three biologically
active double helical structures
of DNA. The others are A- and
B-DNA.
the progression of RFs37,38. The observed pausing of RFs
at transcription bubbles could be due to constraints in the
mobility of the transcription machinery imposed by its
association with the nuclear membrane39. In the case of
a head-on encounter, the slowing down of RFs may also
be caused by the excessive positive superhelicity generated in the downstream DNA by the movements of the
two machineries37,40 (FIG. 2c). In addition to the expected
predominant role of Top1 in resolving the positive supercoils41, a role for Top2 in preventing chromosome fragility at sites of S phase transcription has been recently
described, whereby Top2 was proposed to mediate the
formation of chromatin loops that insulate transcription
units in S phase42.
Natural elements causing RF instability
In addition to the DNA lesions occurring under physiological conditions (such as during hydrolysis or metabolism) or induced by external damaging agents43, the
eukaryotic genome contains numerous natural impediments to replication, such as unusual DNA structures, late
replication zones, DNA-binding proteins and transcription
units. A common feature of these elements is that they
induce the pausing of or completely block the progression
of RFs, increasing the odds for RF breakage events. Several
examples of natural impediments to replication are given
below, and their impact on RF stability and progression
is discussed.
Unusual DNA structures and chromosome fragility. DNA
repeats, such as dinucleotide, trinucleotide, inverted,
mirror and direct tandem repeats, can often undergo
structural transitions that lead to the formation of alternative DNA structures, such as cruciforms, triplex H-DNA,
left-handed Z-DNA and slipped-strand S-DNA, which could
inhibit replication35. The continuation of DNA synthesis
past these elements or the abnormal replication of the
repeats, most often of trinucleotide repeats, has been proposed to lead to their expansion, and this phenomenon
is responsible for many human diseases and hereditary
disorders44–46. Expansion of repetitive elements has also
been associated with chromosomal fragility — a cytogenetic term that describes DNA sequences that show
gaps or breaks, following the partial inhibition of DNA
synthesis47,48, that are often hotspots for chromosomal
rearrangements47. Fragile sites are classified as common
when they are present in all individuals and rare when
they are present in less than 5% of the population. Rare
fragile sites arise as a consequence of repeat expansion and
have been associated with nearly 30 human hereditary
disorders to date46. By contrast, common fragile sites do
not have dinucleotide or trinucleotide repeats, are AT-rich
and are normal components of chromosomes that are
expressed on the inhibition of DNA replication47.
The replication slow zones in yeast are also thought to
represent common fragile sites13,36. Although there is no
AT-rich bias in the replication slow zones, breakage at
these sites is stimulated in the absence of an active ataxia
telangiectasia and Rad3-related protein (ATR) checkpoint
Mec1 in yeast), which is proposed to act by stabilizing the
RFs that are prone to stall at these regions (see also below).
Other replication-stress-sensitive loci have been reported
in yeast and proposed to function analogously with
common fragile sites. Notably, Ty elements and transfer
RNA (tRNA) genes are a big source of genome rearrangements49,50. In yeast strains with reduced levels of
polymerase-α, elevated levels of homologous recombination
(hR)-mediated chromosome translocations frequently
occur at certain Ty elements51. A chromosome region
that contains multiple tRNA genes that are known to stall
RFs is also prone to breakage and translocation events,
particularly in replication checkpoint mutants52.
Exactly what inhibits DNA replication at these fragile
elements, leaving unreplicated or single-stranded
(ss) DNA regions, is unknown. It might be the unusual
conformations that these DNA regions are prone to
adopt47. however, secondary structures should no longer
be favourable as the RF approaches, owing to the positive superhelicity generated in front of the RF53 (FIG. 2a).
hairpins or similar structures could form, however, on the
lagging-strand template in the time window in which this
becomes single stranded, thus interfering with the progression of the lagging-strand polymerase. Preferential
instability of repeats on the lagging strand was indeed
observed in studies performed on replicating plasmids
containing palindromic fragments in both orientations
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Box 3 | Ubiquitin- and SUMO-mediated modifications in DNA replication
Post-translational modification of proteins with ubiquitin or ubiquitin-like proteins,
such as small ubiquitin-related modifier (SUMO), is evolutionarily conserved in all
eukaryotes and affects many signalling networks, including the DNA damage response
(DDR)147. The conjugation machinery involves an E1 activating enzyme, an E2
conjugating enzyme, an E3 ligase to enhance conjugation and/or mediate target
specificity and proteases to deconjugate SUMO from target proteins. Recent
studies provide evidence that ubiquitin-mediated processes contribute to the
replication-stress response in different organisms. The F-box Saccharomyces cerevisiae
protein Dia2 is a Skp1–cullin–F box (SCF) E3 ligase that is important for genome
stability, travels with replication forks (RFs) and regulates RF progression under
conditions of replication stress by interacting with and ubiquitylating replisome
components (mediator of replication checkpoint protein 1 (Mrc1) and chromosome
transmission fidelity protein 4 (Ctf4))148,149. Regulation of S phase by the ubiquitin
proteasome system is also illustrated by the degradation of the DNA replication
licensing factor Cdt1 (ReF. 150) and the minichromosome maintenance 2
(Mcm2)–Mcm7 (MCM) helicase complex22. Other examples include the degradation of
the checkpoint kinase Chk1 after it is activated through phosphorylation by ataxia
telangiectasia and Rad3-related protein (ATR; BOX 1)151,152, the control of the length of
S phase by the E2 ubiquitin-conjugating enzyme UBCH7 (also known as UBE2L3)
(ReF. 153) and the control of repair events by Fanconi anaemia and breast cancer type 1
susceptibility protein (BRCA1) E3 ligases154. The ubiquitylation and sumoylation of the
polymerase clamp proliferating cell nuclear antigen (PCNA) at Lys164 has important
roles in coordinating replication-associated repair events66,67,100,104–106,141, and different
pathways of sumoylation promote RF integrity under conditions of DNA damage68,69.
A new type of PCNA ubiquitylation occurs in both yeast and human cells in response to
a deficiency in the DNA ligase I enzyme that is required to ligate Okazaki fragments
formed during lagging-strand synthesis155, and the findings suggest that distinct
ubiquitylation events on PCNA are required to trigger an efficient DDR caused by
different defects in DNA replication.
Slipped‑strand S‑DNA
A homoduplex DNA formed
between two strands that have
either the same number or a
different number of repeats
(usually triple repeats).
Replication slow zone
A genetically encoded region
that causes slower fork
progression and also tends to
accumulate convergent RFs
and, thus, to represent the
positions of preferential RF
termination.
Ty element
A eukaryotic transposable
element that resembles
retroviruses, with long terminal
repeats at both ends in a
direct orientation. The RNA
intermediate formed by
transcription of the Ty element
is copied as DNA by a reverse
transcriptase encoded by the
Tyb gene of the Ty element. This
DNA copy is then inserted into
a new site in the yeast genome.
Transfer RNA
A small RNA molecule that
transfers a specific active
amino acid to a growing
polypeptide chain at the
ribosomal site of protein
synthesis during translation.
in Escherichia coli 54,55. Another inhibitory factor to replication could be the unusual chromatin structure around
these DNA sequences. Indeed, DNA methylation and the
subsequent heterochromatization of DNA regions around
expanded repeats has been reported56. The binding of
specific proteins to the structure-forming DNA sequence
might also influence the stability of such elements. The
S. cerevisiae hmo1 protein, a high mobility group protein
implicated in chromatin architecture and organization57,
binds to CAG repeat tracts to organize their chromatin
structure and promote tract instability 58.
Transcription and RF stability and dynamics. Occasional
collisions between the replisome and the transcription
machinery are inevitable. Two types of collision can occur:
head-on, whereby the front edge of the RNA polymerase
encounters the components of the RF on the lagging
strand, and co-directional, whereby the components of
the RF on the leading strand encounter the rear edge
of the RNA polymerase (FIG. 2c). Both types of collision
slow down the progression of RFs38. head-on collisions
between the replisome and the RNA polymerase block
replication, leading to RF demise and transcriptionassociated recombination, which may induce genome
instability 19,59. By contrast, it has been suggested that the
RNA transcript in in-line collisions can act as a primer
to continue leading-strand synthesis60. This mechanism
could be particularly relevant when leading strands
encounter damaged DNA, as it would enable the lesion
to be left behind the moving RF, thus precluding RF
stalling and collapse (see below).
Replication blocking sites and termination. Other natural
regions that slow down replication and cause temporal
pausing or nearly complete replication arrest are
RF barriers, at which RFs are purposefully stalled and
replication arrested, and replication termination zones,
at which slowing is topologically imposed by the two
RFs approaching one another (FIG. 2b). RF barriers are
often site-specific and they have been described at telomeres, centromeres, tRNA genes, rDNA arrays (which
code for ribosomal RNAs) and the Schizosaccharomyces
pombe mating type locus. A direct link between RF
stalling and recombination-induced chromosomal
rearrangements was shown when the RF barrier RTS1
was introduced into the S. pombe genome, ensuring
efficient mating-type switching in the fission yeast by
regulating the direction of replication61,62. Recent work
has shown that preventing replication restart at RTS1,
by stabilizing the stalled RF in a manner dependent on
the PCNA-interacting protein Rtf2, allows completion
of DNA synthesis by the converging RF63. In the absence of
Rtf2, slow-moving RFs are established at RTS1 in a
manner that is dependent on the helicase Srs2, which
has been implicated in the regulation of recombination
events during replication64,65. Small ubiquitin-related
modifier (SuMO; Pmt3 in S. pombe; Smt3 in S. cerevisiae), the conjugation of which to S. cerevisiae PCNA
is known to mediate the recruitment of Srs2 to sites
of damage and favour certain recombination events
there66–68, is also required for efficient termination at
the RTS1 element 63.
It is possible that having one of the RFs preferentially blocked by a RF barrier is a general mechanism of
replication termination. Although studies of replication
intermediates in S. cerevisiae did not reveal an important contribution of the sumoylation pathway in stabilizing stalled RFs at certain early origins of replication69,
it is possible that such roles may be site-specific. Indeed,
previous work in S. cerevisiae has shown that yeast with
mutations affecting proteins in the sumoylation pathway, such as the SuMO-conjugating enzyme ubc9, the
SuMO E3 ligase Mms21 (BOX 3) and the cohesin-like
structural maintenance of chromosomes (SMC) complex, Smc5–Smc6, the sumoylation of which depends
on Mms21 (ReF. 70), were not able to efficiently resolve
the cruciform, hemicatenane-like intermediates that
form during the replication of damaged templates69,71
(see below and FIG. 3). Since these structures resemble
termination structures, formed when two replicons
fuse, it has been proposed that sumoylation may be
involved in the termination of replication69,71. The role
of the replication checkpoint in promoting or sensing
replication termination is also not clear. On one hand,
under-replicated DNA, as well as fragility at replication
slow zones and other common fragile sites, is increased
in checkpoint-defective cells13,36. On the other hand, the
under-replication occurring in Cdc14 mutations that are
known to be defective in regulating transition through
mitosis72 is not sensed by the replication checkpoint
and cells progress to anaphase with unreplicated DNA18.
The factors and the regulatory pathways promoting
replication termination remain to be established.
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a
protein A (RPA), and they have a central role in promoting RF stabilization and DNA repair 29,30,73,74. By contrast,
the ataxia telangiectasia mutated (ATM; telomere length
regulation protein 1 (Tel1) in yeast) pathway responds
mainly to double-strand breaks (DSBs) and is important
in promoting the stability of RFs encountering DSB sites
(terminal RFs)75 (BOX 1). When these checkpoint pathways are not functional, unusual structures accumulate
owing to aberrant processing by the nuclease activities
of the stalled, collapsed or terminal RFs73–75. Numerous
studies also document the central role of the Mec1 and
ATR pathways in preventing the expression of fragile
sites13,36,52,76–79.
When S. cerevisiae mutations for the checkpoint proteins Mec1 or Rad53 (BOX 1) are exposed to replication
stress, the replisome dissociates from stalled RFs29,30. This
is thought to be the initial step that then allows the nascent DNA chains to engage in pathological transitions,
such as the formation of reversed RFs 80. It has been
shown that RF reversal leads to topological constraints
that prevent their further regression81 and perhaps generates additional substrates for topoisomerases. Once
reversed, the RFs can be processed to generate the formation of DSBs, which could induce recombination or
long stretches of ssDNA regions73,74,82,83 (BOX 1).
The initial role in promoting RF stability starts with
the RF pausing process itself, which is probably modulated by Mrc1 and Tof1 — two replisome components28
that also function as checkpoint adaptors and influence
the activation of the checkpoint response following
replication stress84–87 (BOX 1). Mrc1 and Tof1 seem to play
an active role in promoting the pausing or stalling of RFs
as, in their absence, the replisome progresses faster than it
is able to synthesize DNA28. Mutations in Mrc1 and Tof1
also increase the fragility associated with expanded triple
repeats79,88. Following the pausing of RFs, it is possible that
the signal is transmitted to Mec1 and Rad53, which prevent replisome disassembly from stalled RFs. The replication checkpoint is also involved in regulating subsequent
steps related to RF restart processes89–92 (see below).
Top1
Top2
Advancing RF
Positive supercoil
Top1 Top2
Top2
Positive supercoil
Precatenane
b
Converging RFs
Positive supercoil
Top1
Top2
Top2
Top2
Precatenane
Precatenane
Top1
Top2
c
Positive supercoil
Advancing RF
Transcription unit
Figure 2 | Topological transitions at the replication fork. a | Unwinding of the duplex
DNA generates a positive supercoil ahead of the replication fork (RF), which is removed
Reviews | Molecular
CellRF,
Biology
mainly by topoisomerase 1 (Top1), but also by Top2Nature
or precatenanes
behind the
the
decatenation of which requires Top2. b | Positive supercoils must be converted into
precatenanes at replication termination. As the RFs approach one another before
fusion, the region available to contain positive supercoils diminishes and precatenanes
form. c | The head-on encounter of a RF with a transcription unit leads to RF demise.
The predominant topoisomerase involved in each situation is shown in red and the
other in yellow.
Homologous recombination
A type of genetic
recombination in which DNA
sequences are exchanged
between two similar or
identical strands of DNA.
The RF and the replication checkpoint
The replication checkpoint stands out as the prime model
of a regulator of replication that affects RF stability.
Intertwined networks of sensors and transducers act to
detect, transmit and amplify the damage or replicationstress signal, which leads to checkpoint activation (BOX 1).
The ATR (Mec1 in yeast) pathway is activated by stalled
RFs with exposed ssDNA regions coated with replication
Types of DNA damage that hinder the RF
Different types of structural and chemical alterations in
DNA can hinder the progression of RFs, leading to RF
demise and the formation of DSBs that could trigger
chromosomal aberrations. The distinct structural constraints imposed by the different lesions elicit the action
of diverse damage tolerance pathways that restore RF
progression or post-replicative repair, as discussed below.
Most of these lesions also trigger the activation of the
damage checkpoint. The roles of the checkpoint pathway
in promoting the repair or damage tolerance events are
complex and not fully elucidated87. The pathways operating during S phase to promote the repair or damage
bypass of different types of lesions are discussed below.
Bulky DNA lesions and damage tolerance pathways.
Exogenous DNA damage, such as by ultraviolet (uV)
irradiation and alkylating agents, and endogenous
processes, such as hydrolysis, which leads to spontaneous DNA depurination, or reactive oxygen species that
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Lesion
Repetitive element
Precatenane
Translesion synthesis
Template switch
Hemicatenane-like structure
Sgs1 and Top3
Error-free bypass
Amplification of the repeat
Figure 3 | Translesion synthesis- and template switch-mediated damage bypass
Nature Reviews
| Molecular
Cell Biology
mechanisms. Replication of damaged templates generates
gaps on
both strands.
A replication fork (RF) with gaps formed on the leading strand owing to repriming events
is shown in the rectangle, in which the structure on the left shows the traditional
representation of the RF with a positive, upstream supercoil and the structure on the
right shows the RF in a precatenane conformation. Damage bypass uses translesion
synthesis (TLS) polymerases, which directly bypass the damage and can often induce
DNA mutations, or a recombination-mediated mechanism called a template switch,
which usually involves the newly synthesized sister chromatid or other sequences with
homology to the single-stranded DNA region present in the gap. Template switching
occurs through a series of events that, in principle, lead to the error-free bypass of lesions,
as indicated in the box on the right. When the region to be copied contains repetitive
elements, slippage and misalignment can lead to repeat contraction or amplification.
An amplification event is highlighted in the box on the left. Top3, topoisomerase 3.
Heterochromatization
The formation of a tightly
packed form of DNA, which
makes the DNA less accessible
to protein factors that usually
bind. Certain DNA elements,
such as centromeres and
telomeres, are heterochromatic.
Bulky lesion
A DNA lesion in which the
nucleotides carry bulky groups.
Methylated DNA and thymine
dimers caused by UV
irradiation are examples.
induce base oxidation and DNA breaks, can lead to the
formation of bulky lesions (for example, DNA adducts) that
stall RFs93. In the semidiscontinuous model of replication
(in which the leading strand is extended in a continuous
manner but the lagging strand contains discontinuities
(FIG. 1b)), blocking the leading replisome dictates that RF
restart occurs at the original site of pausing. This can be
achieved either by using specialized translesion synthesis
(TlS) polymerases that can temporarily replace the replicative polymerase or by switching templates to the newly
synthesized sister chromatid, which is made available
by RF regression mechanisms or by hR-mediated strand
invasion in the homologous duplex 94. hR-mediated
strand invasion involves the assembly and polymerization of Rad51 on the ssDNA that is exposed in proximity
to the lesion. This leads to the formation of the Rad51
presynaptic filament , which can engage in homology
search and invade the homologous region in the
duplex to form a DNA joint, called a displacement loop
(D-loop), which can then be extended through DNA
synthesis (FIG. 3). These two mechanisms — TlS and
template switch — constitute the two main pathways
of damage tolerance, although whether they occur at or
behind the RF is still a matter of debate.
Recent work from several laboratories supports the
conclusion that, in the presence of DNA damage, DNA
synthesis is discontinuous on both strands60,92,95,96. The
leading strand can restart using a primer that the primase
makes on the leading strand, as well as on the lagging
strand95. Alternatively, the mRNA transcript can be used as
a primer by the leading-strand polymerase when it encounters the RNA polymerase of a transcription unit during inline collisions60,97. These mechanisms will leave the bulky
lesion behind the RF, concealed in a gap, both on the
leading and lagging strands. Gaps can later be filled in by
TlS polymerases or template switch mechanisms, as mentioned above (FIG. 3). In this view, these damage tolerance
pathways take place behind the RF.
The damage replication checkpoint influences both
branches of damage tolerance. The RAD9–RAD1–huS1
(also known as 9-1-1) damage checkpoint facilitates the
recruitment of the TlS polymerase Polζ in S. cerevisiae
and DinB (also known as Mug40) in S. pombe to sites of
damage89,90, and damage-induced mutagenesis is partially
dependent on the damage checkpoint 98–100. S. cerevisiae
Rad53 and phosphorylation of the 9-1-1 complex on
Rad9 by the ATM- and ATR-related Rad3 checkpoint
kinase in S. pombe100 also promote error-free-mediated
gap filling 91,100.
Genetic studies conducted in yeast have already
established that error-free-mediated damage bypass
by template switching occurs by two main pathways:
one mediated by Rad18–Rad5–Mms2–ubc13, referred
to as error-free post-replication repair (PRR), and the
other one mediated by Rad51–Rad52 (ReFS 101–103).
These pathways cooperate to promote the bypass of the
lesion through sister chromatid pairing, which leads
to cruciform structures that resemble hemicatenanes68.
Monoubiquitylation and polyubiquitylation of PCNA at
the highly conserved residue lys164 (ReF. 104) differentially affects the two pathways of damage tolerance. Rad6and Rad18-dependent monoubiquitylation promotes TlS
in both yeast and mammalian cells105,106, whereas ubc13–
Mms2 and Rad5-dependent PCNA polyubiquitylation
in yeast through lys63-linked chains is required for
error-free PRR104,107,108 and template switch-mediated
sister chromatid junction formation68. In yeast, PCNA
is also sumoylated104 and biochemically these modifications occur independently of each other 66,67,104. however,
in the absence of PCNA sumoylation, the ability of the
error-free PRR pathway to promote template switching
through sister chromatid junctions is impaired68, suggesting that PCNA sumoylation favours the damage
bypass of lesions by enabling the use of factors belonging
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DNA adduct
A piece of DNA that is
covalently bonded to a
chemical.
Homologous duplex
A DNA duplex that shows
homology with another
DNA region or sequence and
usually contains a DSB.
Presynaptic filament
A nucleoprotein filament
consisting of Rad51
molecules bound to ssDNA.
Hemicatenane
A cruciform junction of two
dsDNA molecules, in which
one of the strands of one
duplex passes between the
two strands of the other
duplex, and vice versa.
Microhomology
Refers to homologous
sequences that are extremely
small, usually just a few base
pairs long.
to the error-free branch of PRR. This interpretation is
in agreement with genetic data in budding and fission
yeasts, in which a lack of PCNA sumoylation was
shown to suppress the damage sensitivity of error-free
PRR mutants66,67,100. The exact mechanism underlying
this phenomenon is unknown but, besides influencing
the interacting partners of PCNA, sumoylation might
affect the chromatin structure or the accessibility of
factors such as Rad18 to sites of DNA lesions. In support of this, a recent study has found that the chromatin
remodelling factor inositol-requiring protein 80 (Ino80)
is important for efficient PCNA ubiquitylation following
methyl methanesulphonate treatment, the recruitment of
Rad18 and Rad51 to damaged RFs and the formation
of sister chromatid junctions during replication of
damaged templates109.
The sister chromatid junctions formed through
template-switch processes are resolved by the action of
the RecQ helicases Sgs1 (in yeast) and Bloom syndrome
protein (BlM; in humans), together with the topoisomerase Top391,110,111 (FIG. 3). Sumoylation mediated by
ubc9 and the SuMO E3 ligase Mms21, and the Mms21associated SMC complex, was shown to counteract the
accumulation of these structures69,71,112. understanding
the factors contributing to the timely resolution of these
structures is important as, in the absence of Sgs1 (or
BlM), Top3 and other regulatory activities, the cruciform
structures may become targets for endonucleases, leading
to DSB formation and excessive recombination (known as
hyper-recombination).
As precatenane formation is coordinated with replication it is possible that, together with cohesion, precatenanes
facilitate the interaction between sister chromatids and,
thus, subsequently also facilitate template switching
(FIG. 3). Template switching is mostly an error-free mechanism, but the replication of DNA fragments containing
repetitive elements often leads to misalignments and the
production of repeat rearrangements113 (FIG. 3). Indeed,
factors implicated in promoting template switching,
such as Rad6, Rad5 and Sgs1 (ReF. 68), were also shown
to increase the instability of repeated sequences88,114. RFs
containing hairpins or other secondary DNA structures
can be preferentially contained in chromatin loops or in
higher-order DNA conformations. Pairing of the initially
blocked nascent chain with the sister chromatid (FIG. 3), or
with a strand from a distant RF that contains microhomology
elements and is brought nearby by chromatin looping
events, could lead to genome rearrangements115,116. Recent
studies in budding and fission yeast have shown that RF
arrest in repeated sequences of nearby inverted repeats
can lead to a switch of templates that generate dicentric
chromosome intermediates, which, on segregation, lead to
breakage and further chromosome rearrangements117,118.
understanding how genome architecture affects
replication-associated repair events is a fascinating topic
in chromosome and cellular biology.
Interstrand cross links and their repair. Covalent links
between complementary DNA strands completely
block RF progression by precluding strand separation.
Interstrand cross links (ICls) are widely generated by
cancer chemotherapy and are also formed by cellular
metabolites119. In mammalian cells, the repair of ICls
involves the interplay of the Fanconi anaemia pathway,
hR and TlS polymerases120. Cells from individuals
afflicted with Fanconi anaemia, a rare disorder characterized by congenital abnormalities and an increased
incidence of cancer, exhibit chromosomal fragility and
are notoriously hypersensitive to ICl agents121. Indeed,
the Fanconi anaemia pathway prevents the formation
of replication-associated DSBs122. Fanconi anaemia proteins can be classified into three groups. Group I includes
eight Fanconi anaemia proteins that constitute the core
complex. This complex has ubiquitin ligase activity 121,123
and ubiquitylates FANCI and FANCD2, which constitute the group II proteins124 (FIG. 4). In addition, Fanconi
anaemia-associated protein of 24 kDa (FAAP24), a protein that shares homology with the XPF family of endonucleases, interacts with the FANCM helicase of the
Fanconi anaemia core complex, and is required for full
levels of FANCD2 ubiquitylation in S phase or after
damage125. ubiquitylation of FANCI–FANCD2 is essential for its ability to form foci on damaged chromatin,
where other recombination proteins, such as RAD51,
breast cancer type 1 susceptibility protein (BRCA1),
and BRCA2 (also known as FANCD1), are recruited124.
BRCA2, together with its interacting protein FANCN
(also known as PAlB2) and the BRCA1-associated
DNA helicase FANCJ (also known as BRIP1)126 are
group III Fanconi anaemia proteins. These proteins do
not affect FANCD2 ubiquitylation, but contribute to hR
repair 127,128 (FIG. 4).
The exact role of Fanconi anaemia proteins in repair
is not yet understood, but genetic evidence suggests that
they might act to create an appropriate substrate for hR
or TlS129. This substrate may be reversed RFs, as FANCM
was reported to have RF remodelling activity that can convert a RF into a four-way junction130–132. In S. cerevisiae,
the helicase activity of the FANCM orthologue mutator
phenotype protein 1 (Mph1) was shown to promote the
accumulation of recombination intermediates that require
the Smc5–Smc6 complex for resolution133.
Recent studies using a cell-free system based on
Xenopus laevis egg extracts analysed the replication of a
plasmid containing a site-specific ICl134 (FIG. 4). In this
system, the two RFs converge at the cross link, with both
leading strands initially stalling 20–40 nucleotides away
from the lesion, perhaps owing to topological constrains.
One leading strand is then extended to within one nucleotide of the ICl, and dual incisions are made on both sides
of the ICl on the parental DNA leading strand that contains the RF that stalled 20–40 nucleotides away from the
lesion. Repair then occurs through TlS-mediated bypass
of the lesion on the strand on which further elongation took place and by hR on the other strand134 (FIG. 4).
ubiquitylated FANCI and FANCD2 were shown to be
required for the nucleolytic incision near the ICl and for
the TlS step135.
The two RFs converging at the ICl site resemble
the situation that occurrs at replication termination.
Whether RF reversal is actively prevented by uncoupling
the leading and the lagging strands of the converging
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a
b
S phase damage
ATR ATRIP
ICL
Replisome
FANCL
FANCA FANCB FANCC FAAP24
FANCE FANCF FANCG FANCM
RF advance
Uncoupling of leading
and lagging strands
FANCI FANCD2
P
P
P
P
Incision (topoisomerases and
other nucleases?)
P
FANCA FANCB FANCC FAAP24
FANCL
FANCE FANCF FANCG FANCM
FANCI FANCD2
E2
Ub
P
P
P
P
P
FANCA FANCB FANCC FAAP24
FANCL
FANCE FANCF FANCG FANCM
FANCI FANCD2
Ub
Translesion synthesis
Resection
RAD51 RAD52 RAD54
FANCJ BRCA1
FANCN BRCA2
Nucleotide excision repair
Homologous recombination
Ub
Figure 4 | Regulation of the Fanconi protein complex in response to DNa damage and a model for interstrand
cross link repair. a| S phase damage activates the ataxia telangiectasia and Rad3-related protein (ATR) checkpoint,
Nature
| Molecular
Cell core
Biology
which phosphorylates several Fanconi anaemia proteins. The different Fanconi subunits
are Reviews
indicated:
group I (the
complex; shown in the rectangle) and group II (FANCI and FANCD2; the ID complex). FANCM and Fanconi anaemiaassociated protein of 24 kDa (FAAP24) have enzymatic activities. ATR-dependent phosphorylation of Fanconi
anaemia proteins induces the ubiquitin ligase activity of the core complex and leads to robust ubiquitylation of FANCI and
FANCD2. This is required for their association with chromatin in foci that contain several other recombination proteins:
RAD51, RAD52, RAD54, and breast cancer type 1 susceptibility protein (BRCA1), as well as the FANCN (also known as
PALB2), FANCJ (also known as BRIP1) and BRCA2 (also known as FANCD1) Fanconi subunits of group III (shown in blue).
b| A model of interstrand cross link (ICL) repair, in which the replication forks (RFs) converge at the cross link. One leading
strand is then extended to within one nucleotide of the ICL. The topological constraints are relieved by nicking events,
which cause incisions and release the two sister strands. Repair then occurs through the translesion synthesis-mediated
bypass of the lesion, followed by nucleotide excision repair-mediated excision of the lesion on one strand, and resection
and homologous recombination repair of the other strand. ATRIP, ATR-interacting protein; Ub, ubiquitin.
Dicentric chromosome
intermediate
An unstable chromosome
that has two centromeres.
DNA decatenation
The unknotting of catenated
structures.
Camptothecin
A natural alkaloid that inhibits
TOP1. Camptothecin
analogues are often used in
cancer chemotherapy.
RFs134 or by releasing the topological constraints caused
by the ICl through incision events mediated by topoisomerases or specialized nucleases remains unknown.
It is also possible that topoisomerases might need to
collaborate with recombinases for the final resolution of
the recombination structures formed during ICl repair.
Interestingly, the Fanconi-associated factor BRCA1 was
shown to promote DNA decatenation136. This is mediated
through its interaction and colocalization with TOP2A
(BOX 2), the ubiquitylation of which, mediated by the E3
ubiquitin ligase activity (BOX 3) of BRCA1, correlates with
higher DNA decatenation activity 136.
The replication checkpoint directly regulates the
Fanconi anaemia pathway: both FANCD2 and FANCI
are ATR targets137 and their phosphorylation by ATR
affects the ubiquitylation of this complex (FIG. 4). A feedback loop between FANCM and the checkpoint effector
kinase ChK1 (also known as ChEK1) (BOX 1) stabilizes
these proteins from degradation by the proteasome
under conditions of DNA damage138. Thus, a complex
network of checkpoint and ubiquitin-mediated signalling and repair activities may be required to facilitate ICl
repair and prevent the genomic instability associated with
Fanconi anaemia.
DNA–protein cross links and DSB repair. DNA–protein
complexes are induced by oxidative damage or uV irradiation. Anticancer camptothecins, such as topotecan or
irinotecan, lead to reversible TOP1–DNA cleavage complexes, in which topoisomerases are covalently linked
to the broken DNA strands139. It has been proposed that
camptothecin treatment arrests advancing RFs through
the accumulation of positive supercoils ahead of the
replication machinery 140. Whether DSBs arise as a consequence of topological strain or the dissociation of the
replisome when it encounters a topoisomerase–DNA
complex (a nick) remains unknown. The repair of DSBs
is not restricted to S phase and, therefore, an in-depth
description of the mechanisms involved is not addressed
here, but we invite readers to other reviews141,142.
In addition to the DSBs formed by collisions of RFs
with covalent DNA–protein complexes, RFs can encounter
DSB sites or eroding telomeres. In a recent study in yeast
it was shown that RFs irreversibly resolve at the break site,
and that the Tel1–ATM checkpoint acts together with the
Sae2 and the Mre11 nucleases to prevent the formation
of cruciform structures at such terminal RFs75 (BOX 1).
Arrest of RF movement on encountering a DSB has also
been observed using an SV40 cell-free replication system,
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analysing the collision between a RF and a Top1-cleavable
complex143. The termination of the RF that encounters the
DSB does not impede the progression of the sister RF that
is generated during replication initiation at the same replicon, suggesting that sister RFs can be uncoupled during
replication75. A nearby DSB also triggers origin firing,
even the firing of origins that are dormant under normal
conditions of replication75.
Concluding remarks and perspectives
Studies conducted in recent years have helped identify
and establish the roles of several factors in activating the
replication and damage checkpoint pathways following
replication stress, promoting RF restart and progression
or facilitating numerous replication-associated repair
processes. Additional regulatory pathways involving ubiquitin and SuMO modifications now stand out, together
with the regulatory pathways mediated by checkpoints
and CDKs, as important coordinators of repair events and
DNA transactions. how these factors and regulatory pathways affect topology and chromatin structure, in addition
to influencing protein interactions, cellular distribution, or
target stability, remain largely unsolved questions. Much
has been learnt about checkpoints and their targets, as well
as about other factors with important roles in replication
and genome stability, such as RecQ proteins and DNA
topoisomerases. however, many questions are arising and
many still remain to be answered. Clearly, these fascinating
enzymes will continue to keep scientists mesmerized and
the replication and repair fields exciting.
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Genetic screens in yeasts and vertebrates and enzymological studies have been extended and combined
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causes include altered replication kinetics, the formation
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Acknowledgements
We thank all members of our laboratories for helpful discus‑
sions. The work in D.B’s laboratory is supported by
the European Research Council grant 242928 and the
Associazione Italiana per la Ricerca sul Cancro. The work in
M.F’s laboratory is supported by grants from Telethon,
the Associazione Italiana per la Ricerca sul Cancro and the
European Community.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
uniProtKb: http://www.uniprot.org
ATM | ATR | BLM | BRCA1 | BRCA2 | Cdc6 | Cdc7 | Cdc45 |
Csm3 | Dbf4 | DinB | FAAP24 | FANCD2 | FANCI | FANCJ |
FANCM | FANCN | Hmo1 | Mec1 | Mms21 | Mph1 | Mrc1 |
PCNA | Pmt3 | Rad51 | Rad53 | Rtf2 | Sgs1 | Smt3 | Tel1 | Tof1 |
Top1 | Top2 | Ubc9
FURTHER INFORMATION
Dana branzei’s homepage: http://www.ifom-ieo-campus.it/
research/branzei.php
marco foiani’s homepage: http://www.ifom-ieo-campus.it/
research/foiani.php
all liNks aRe acTive iN The oNliNe pDF
VOluME 11 | MARCh 2010 | 219
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